RSV represents a major health problem, worldwide. In the United States alone, there are currently approximately 250,000 new born infants and children per year who may develop severe or fatal RSV disease. RSV is the major viral cause of severe pediatric lower respiratory tract diseases, such as pneumonia and bronchiolitis, worldwvide. It also results in a high rate of morbidity and mortality in infants or young children with cardiopulmonary disease or an immunodeficiency.
In addition to the childhood population at risk (McIntosh and Chanock (1990) Virology, 2nd edn. (Fields and Knipe, eds) Raven Press, Ltd., New York, pp. 1045-1072), there is a considerable and increasingly large population of immunosuppressed adults at risk due to the increasingly widespread application of organ transplant, cancer/leukemia therapies such as bone marrow transplantation and the proliferation of HIV infections in the homosexual population. HIV is now the leading killer of homosexuals in the 25-40 year age group, in the U.S.A. The aged, who represent a growing population in developing countries, also are at risk due to immune deficiencies resulting from their acing immune systems, and RSV can be endemic in nursing home populations, particularly during the Winter season.
Whilst antibiotic therapy of bacterial infection has been successful in many diseases, few antibiotics are available for therapy of viral infections and none are currently available for effective treatment of RSV infection. However the severity of viral infections is usually also correlated with the immune status of the patient. For example, there is a correlation between levels of maternal IgG antibodies to RSV and the resistance of infants to infection during the first months of life, when the risk of severe disease is greatest (Ogilvie, et al., J. Med. Virol. 7:263, 1981). Pooled human gamma globulin with high titer RSV neutralizing antibodies or RSV neutralizing murine monoclonal antibodies can protect small animals from pulmonary infection with RSV and, when administered therapeutically, can be effective in small animals and primates at the height of RSV infection (Walsh, et al., Infection and Immunity, 43:756, 1984; Prince, et al., J. Virol., 55:517, 1985; Prince, et al., Virus Research, 3:193,1985 Prince, et al., J. Virol., 61:1851, 1987; Hemming, et al., J. Inf. Dis., 152:1083, 1985). Pooled human IgG containing RSV neutralizing antibodies has also been used clinically, to therapeutic effect, in a study of serious RSV disease in infants and young children (Hemming, et al., Antimicrob. Agnts. Chemotherap., 31:1882, 1987). However the use of pooled human sera for the treatment of RSV infection has several drawbacks. Availability is limited. Batches are not reproducible. Titers are 100 to 1,000 fold lower than for monoclonal antibody titers and the risk of iatrogenic infection is always present when using human serum, due to the variable resistance of microorganisms to the sterilization procedures utilized.
An RSV vaccine for active immunization, if available, could not be utilized for the treatment of newborn babies with immature immune systems or patients who are immunosuppressed. In patients where prophylactic passive immunotherapy is required, as a result of a more chronic form of disease, current therapy is mediated via periodic intravenous inoculation of human IgG prepared from pooled plasma. This type of therapy, due to the low titers of neutralizing anti-RSV antibodies, involves a large quantity of globulin (e.g., 0.75 gm per kg) and consequently requires administration intravenously, in a clinic or hospital, over a lengthy period (2 to 4 hours), on a monthly basis during the high risk months (fall, winter and early spring).
The neutralizing component of human anti-RSV antibody preparations, derived from pooled human plasma, is only a minor fraction of the total antibody present. The development of mouse monoclonal antibody technology thus provided cloned neutralizing antibodies of greater specific activity than the pooled human plasma preparations. However problems resulting from immune responses to the mouse antibodies, in human patients, have precluded the general application of these preparations for passive immunotherapy in humans. The development of human monoclonal antibodies to RSV has been thwarted, until recently, by the unsuccessful adaptation of monoclonal technology to the human system. Human hybridomas and immortalized EBV transformed B-lymphoblastoid cell lines, as well as mouse/human hybridomas are generally unstable antibody producers, even after multiple cloning steps. The cloning and expression of human monoclonal antibodies, in E. coli utilizing phage (Huse et al., Science 246:1275-1281, 1989; Clackson et al., Nature 352:624-628, 1991; Barbas et al., Proc. Natl. Acad. Sci. (USA): 88:7978-7982, 1991), has obviated this problem. RSV-specific human monoclonal antibody is now available with a 100 to 1000-fold higher concentration of specific antibody than pooled plasma preparations. The use of these human monoclonal antibody preparations will correspondingly decrease the volume of antibody preparations required for prophylaxis or therapy by the same order of magnitude. Effective doses of monoclonal antibody may now be administered intramuscularly (i.m.), thereby reducing the period of time required. Prophylaxis in new born babies or infants can now be performed at home, as opposed to in the clinic or hospital, reducing inconvenience and eliminating the risk of hospital acquired RSV disease. This is in addition to the inherent reduction in batch to batch variation of monoclonal antibody preparations and the reduction of the danger of iatrogenic infections when compared to pooled human globulin. In fact, the reduced volumes of antibody preparations required for therapy will allow, in general, treatment of patients with RSV disease by administration of antibodies intramuscularly. Aerosol therapy is another form of treatment made possible as a result of the increased specific activity of monoclonal antibodies, and is also associated with a decrease in the amount of antibodies required. This type of therapy is highly efficient due to the introduction of antibodies directly to the site of infection in the lungs. The neutralizing ability of Fab fragments of the RSV monoclonal antibodies in vivo, by aerosol application or systemic therapy, has been well demonstrated.
Neutralizing epitopes on the RSV virus are mainly confined to the major surface antigens: the F glycoprotein (viral fusion) and G glycoprotein (viral attachment). Antiserum prepared against either glycoprotein F or glycoprotein G may neutralize RSV with high efficiency (Walsh, et al., J. Gen. Microbiol., 67:505, 1986). However antibodies to glycoprotein F are more frequently neutralizing for RSV. Antiserum to glycoprotein F also inhibits fusion of RSV-infected cells to neighboring uninfected cells. For therapeutic purposes, antibody preparations should neutralize a wide range of RSV isolates, including those of both antigenic subgroups. There are two antigenic subgroups of RSV, A and B, which are each present at all times in the population but which vary in proportion at any given time. Subgroups A and B are 50% related in glycoprotein F at the DNA sequence level, but appear to be more highly related in the neutralization epitope regions. In contrast, subgroups A and B are only 10% related in glycoprotein G (McIntosh and Chanock, supra). During the last several years, the efficiency of topical immunotherapy for RSV infection has been increased by two modifications of previous methodology. First, a mixture of RSV F immune monoclonal antibodies directed at the major conserved neutralization epitopes on this glycoprotein was shown effective in topical immunotherapy of RSV infection in the cotton rat. Second, delivery of RSV polyclonal antibodies directly into the lungs in a small particle aerosol (less than 2 μm) was also effective therapeutically. The use of monoclonal antibodies should decrease the amount of IgG required for therapy by at least 2 orders of magnitude. In other studies in cotton rats, parainfluenza virus type 3 (PIV3) antibodies were also shown to be therapeutic against PIV3 when administered directly into the respiratory tract. This form of topical immunotherapy has general application for respiratory viral pathogens causing disease in the cells lining the lumen of the lower respiratory tract.
Humanized mouse monoclonal antibodies (MAb), due to the contribution of the grafted mouse CDR sequences, retain a significant proportion of mouse sequence, representing 25-30% of the V-regions. There is no evidence to suggest any relationship between the mouse RSV 19 (Taylor et al., Immunology 52:137-142, 1984) and published human antibody V-region CDR sequences (Winter et al., Eur. J. Immunol. 21:985-991, 1991) and hence repeated administration of humanized mouse MAb, as a consequence of the surface location of the CDR regions on the antibody molecule, is likely to result in a human anti-mouse MAb (HAMMA) response. This response would then preclude further therapeutic use of the humanized mouse MAb and in particular preclude any use of these humanized mouse MAb sequences for antibody gene therapy, in which case the therapy could not be withdrawn and might adversely affect the health of the patient. HAMMA responses are common in patients given conventional systemic therapy with mouse MAb, resulting in up to 50% of patients responding after the first dose and up to 95% of patients responding after the second dose. The use of pooled human gamma globulin has been universal for prophylaxis in hepatitis and for treatment in hepatitis, Junin virus induced hemorrhagic fever and RSV infection, with no side effects severe enough to preclude this form of passive immunization. Hence, by inference, the application of a human monoclonal Fab to this form of therapy should have no serious consequence such as that induced by the HAMMA response to mouse antibody or fragments thereof.
For long term prophylaxis of RSV infection in immunosuppressed patients or newborn infants who lack an intact immune system, it would be preferable to apply an immunoglobulin preparation for passive immunization which includes more than one neutralizing epitope on the RSV F glycoprotein. This is due to the mutation rate of the RSV F glycoprotein for any single neutralization site being in the range 10−4 to 10−5, the rate for two neutralization sites being thus 10−8 to 10−10, for three neutralization sites being 10−12 to 10−15 and so on. Administration of anti-RSV antibodies or fragments thereof, over a significant period of time in multiple patients or as multiple periods of treatment in a single patient, would create a significant selective pressure for the development of escape mutants. Hence, to counteract this selective pressure, the inclusion of antibodies or fragments to two or more neutralization epitopes is preferable in any preparation to be used for passive immunization. However, prior to the present invention, only one other neutralizing human monoclonal RSV antibody or fragment thereof, was known. The human Fab RSV 19 of Barbas et al. (Proc. Natl. Acad. Sci. (USA) 89:10164-10168, 1992), included in PCT International Publication Number WO 94/06448, has an amino acid sequence completely unrelated to those of the anti-RSV antibodies of the present invention. More important, the RSV19 human Fab binds to an unrelated neutralization epitope on the RSV F glycoprotein epitope, representing the “B” epitope or antigenic site, recognized by the mouse MAb 1269 (Taylor et al., Immunology 52:137-142, 1984). Hence the uniqueness of the anti-RSV antibodies of the present invention and the human Fab RSV19, in both aa sequence and epitopic site, has important implications for the design of immunotherapeutic vaccines or modalities for the treatment of RSV disease.